U.S. patent application number 15/908105 was filed with the patent office on 2018-09-06 for biologically-based control methods for insect pests.
The applicant listed for this patent is Foresight Science & Technology, Inc., The United States of America, as represented by the Secretary of Agriculture, The United States of America, as represented by the Secretary of Agriculture. Invention is credited to Satya P. Chinta, Robert K. Vander Meer.
Application Number | 20180249708 15/908105 |
Document ID | / |
Family ID | 63357208 |
Filed Date | 2018-09-06 |
United States Patent
Application |
20180249708 |
Kind Code |
A1 |
Vander Meer; Robert K. ; et
al. |
September 6, 2018 |
BIOLOGICALLY-BASED CONTROL METHODS FOR INSECT PESTS
Abstract
The present invention provides novel compositions and methods
for using the insect neuromodulator tyramine and/or one or more
derivatives thereof delivered to insect populations to control
certain aspects of such populations. The amount of tyramine and/or
one or more derivatives thereof delivered on a per insect basis is
significantly greater than a naturally-occurring amount of tyramine
normally found in insects and causes deleterious effects including
mortality of certain insect life forms and disruption of the
reproductive capacity of others in the population.
Inventors: |
Vander Meer; Robert K.;
(Newberry, FL) ; Chinta; Satya P.; (Gainesville,
FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary of
Agriculture
Foresight Science & Technology, Inc. |
Washington
Hopkinton |
DC
MA |
US
US |
|
|
Family ID: |
63357208 |
Appl. No.: |
15/908105 |
Filed: |
February 28, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62465503 |
Mar 1, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01M 1/2011 20130101;
A01N 25/006 20130101; A01M 2200/011 20130101; A01N 33/10 20130101;
A01N 33/10 20130101; A01N 25/006 20130101 |
International
Class: |
A01N 33/10 20060101
A01N033/10; A01N 25/00 20060101 A01N025/00; A01M 1/20 20060101
A01M001/20 |
Claims
1. A composition for controlling a target population of social
insects, the composition comprising an effective amount of a
compound selected from the group consisting of: tyramine, one or
more derivatives of tyramine, and combinations thereof, wherein the
effective amount is sufficient to control a target population of
social insects.
2. The composition of claim 1, wherein the effective amount of the
compound is present in a concentration is at least about 0.0001% on
a weight per volume basis.
3. The composition of claim 1, wherein the effective amount of the
compound is present in a sufficient quantity such that it is
delivered to the target population of insects at least about 0.01
micrograms per insect in the target population of social
insects.
4. The composition of claim 1, wherein the effective amount of the
compound is present in a sufficient quantity such that it is
delivered to the target population of insects at least about 50
times an amount of tyramine naturally occurring within an insect in
the target population of insects.
5. The composition of claim 1, wherein the compound is in a form
selected from the group consisting of: substantially pure, solid,
liquid, semi-solid, spay, powder, granule, tablet, gel, cream,
lotion, and combinations thereof.
6. The composition of claim 1, further comprising a component
selected from the group consisting of: liquid diluent, solid
diluent, surfactant, additive, preservative, and combinations
thereof.
7. The composition of claim 1, further comprising a component
selected from the group consisting of: a phagostimulant, an
ant-attractant component, an ant-toxic component, an ant-ingestible
component, and combinations thereof.
8. A method of controlling a target population of social insects,
the method comprising delivering an effective amount of a compound
selected from the group consisting of: tyramine, one or more
derivatives of tyramine, and combinations thereof, wherein the
effective amount is sufficient to control a target population of
social insects.
9. The method of claim 8, wherein the target population of insects
is selected from the genera consisting of: Monomorium, Myrmicaria,
Solenopsis, Megalomyrmex, Trachymyrmex, Cyphomyrmex, Tetramorium,
Pheidole, Myrmica, Crematogaster, Pogonomyrmex, Atta, and other
genera within the ant sub-family Myrmicinae.
10. The method of claim 8, wherein the target population of insects
comprises Solenopsis invicta.
11. The method of claim 8, wherein the effective amount of tyramine
is from about 0.01 microgram to about 2.0 micrograms of the
compound per insect in a population of insects.
12. The method of claim 8, wherein the compound includes tyramine
without any tyramine derivatives.
13. The method of claim 8, wherein the compound includes tyramine
and its derivatives.
14. The method of claim 8, wherein the compound includes tyramine
derivatives in the absence of tyramine.
15. The method of claim 8, wherein the tyramine composition is in a
form selected from the group consisting of: solid, liquid,
semi-solid, spay, powder, granule, tablet, gel, cream, lotion, and
combinations thereof.
16. The method of claim 8, wherein the tyramine composition further
comprises a component selected from the group consisting of: an
ant-attractant component, an ant-toxic component, an ant-ingestible
component, and combinations thereof.
17. The method of claim 8, further comprising deploying the
compound in a bait station.
18. A kit for carrying out the method of claim 8, the kit
comprising a first container means containing the compound;
optionally other container means comprising a solution, diluent, or
applicator for the compound; and written information including
procedures for applying the compound.
19. A method of controlling a population of fire ants, the method
comprising delivering a compound comprising an effective amount of
tryamine and/or at least one of its derivatives to the population
of fire ants, wherein the amount of tyramine delivered on a per
fire ant worker basis is at least about fifty times greater than an
average amount of naturally-occurring tyramine within fire ant
workers in the population of fire ants.
20. The method of claim 19, wherein the amount of tyramine and/or
its derivatives is combined with an ant-attractant compound to
create an ingestible bait composition.
21. The method of claim 20, wherein the ingestible bait composition
further comprises a phagostimulant.
22. The method of claim 19, wherein the amount of tyramine and/or
its derivatives delivered on the per fire ant worker basis is at
least about one thousand times greater than the average amount of
naturally-occurring tyramine within fire ant workers in the
population of fire ants.
23. The method of claim 19, wherein the amount of tyramine and/or
its derivatives delivered on the per fire ant worker basis is from
about one thousand times to about ten thousand times greater than
the average amount of naturally-occurring tyramine within fire ant
workers in the population of fire ants.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 62/465,503, filed Mar. 1, 2017, the content of
which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The disclosed invention relates generally to compositions
and methods for controlling pest social insect populations using
naturally-occurring or synthetic biogenic amine compounds or
derivatives thereof. More specifically, the invention relates to
compositions and methods for exposing targeted ant colonies to such
biogenic amines for the control and elimination of the
colonies.
BACKGROUND OF THE INVENTION
[0003] Ants comprise 5% of the world's 100 worst invasive alien
species and 28% of land invertebrates (see Lowe S., et al., (2000).
100 of the World's Worst Invasive Alien Species A selection from
the Global Invasive Species Database. Published by The Invasive
Species Specialist Group (ISSG) a specialist group of the Species
Survival Commission (SSC) of the World Conservation Union (IUCN),
12pp). It has been estimated that there are over twenty thousand
ant species occupying every ecological niche as part of the
structure of ecological communities (see Holldobler and Wilson, The
Ants. Harvard University Press, Cambridge, 1990; Smith et al., Cold
Spring Harb. Protoc. 4:1, 2009), and over 12,500 currently
described (antbase.org, 15 Jan. 2010). However, some of the ant
species, especially those moved through human activities to
non-native locations, have become serious pests (see Lessard et
al., Ecology 90:2664, 2009; Porter and Savignano, Ecology 71:2095,
1990). The Myrmicinae subfamily, for example, is the largest of the
21 extant subfamilies in the ants (Hymenoptera: Formicidae)
(Bolton, Mem. Am. Entomol. Inst. 71:1, 2003). The Myrmicinae
subfamily includes about 140 genera within the group, including
species with a functional sting (such as, for example, ants in the
genus Solenopsis, one of which is S. invicta, known as the "red
imported fire ant," and Myrmica rubra, also known as the European
fire ant or common red ant), and fungus-growing ants (of the tribe
Attini, e.g., leafcutter ants (genus Atta and genus Acromyrmex).
Collectively and individually, these and other ant species are
considered pests in commercial, agricultural, and residential
settings, causing significant damage and injury to crops and
livestock and a host of other economic sectors, as well as
potential medical issues (e.g., humans and animals can develop
hypersensitivity to fire ant venom that may result in anaphylactic
shock and death).
[0004] Global commerce ensures that these invasive pest species,
including social insects such as various ant species (e.g.,
imported/invasive fire ants) will be distributed to compatible
habitats throughout the world. For example, it is estimated that
imported fire ants currently infest hundreds of millions of acres
in southern tier states and Puerto Rico and are spreading
northward. As is the case for most exotic species, invasive fire
ants were introduced without most of their natural enemies in their
native South American range. As a consequence, fire ant populations
in the United States are estimated to be about 5-10 times denser
than in South America. In recent decades S. invicta, for example,
has changed from an invasive pest ant in the United States to a
global problem, with infestations occurring in Australia, Taiwan,
Mainland China, Mexico, and many Caribbean Island countries. This
invasive ant is estimated to be responsible for almost $7 billion
annually in damage repair, medical care, and control costs in the
United States alone. Broad ranging economic sectors are impacted by
fire ants including households, electric and communication
services, outdoor recreation areas, agriculture, schools, and other
areas.
[0005] Limited options exist for biologically-based methods of
controlling or eliminating invasive and native pest ant populations
in urban and rural environments. These limited options are due, in
large part, to reliance on conventional pesticides for control,
which in turn creates environmental and health risks associated
with such pesticides. The use of broad-spectrum, persistent
insecticides has significant drawbacks in that the chemicals also
kill other beneficial insects and may contaminate surface and
ground water due to runoff as well as create other environmental
concerns.
[0006] There thus exists an industrial, commercial, and public
ongoing need to develop efficacious, novel, and safe compositions
and control methods to alleviate these problems, particularly for
the control of invasive fire ants, as well as other invasive ants
and insects, which are also cost-effective and easily adaptable for
widespread use.
SUMMARY OF THE INVENTION
[0007] To address the challenges in developing biologically-based
compositions and methods of controlling insect populations, the
present invention accordingly provides novel compositions and uses
for the insect neuromodulator tyramine and/or its derivative(s)
delivered to insect populations to control certain aspects of those
populations. The aspects include, for example, fecundity,
reproduction, wing loss, worker lifespan, among others. An aspect
of the invention provides compositions for controlling a target
population of social insects. The compositions include an effective
amount of a compound selected from the group consisting of
tyramine, one or more derivatives of tyramine, and combinations
thereof. The effective amount in the composition is sufficient to
deliver prescribed dosages to the individual insects in the
population and control a target population of social insects. In
another aspect, the invention provides methods of controlling a
population of insects, for example, fire ants. The method includes
delivering an amount of tryamine and/or its derivative(s) to the
population of insects. The amount of tyramine and/or derivative(s)
thereof delivered on a per fire ant basis is greater than a
naturally-occurring amount of tyramine normally found in the
insects (e.g., within the brains of certain fire ants in the
population of fire ants to be controlled). It was surprisingly
discovered that delivering such amounts of tyramine and/or its
derivative(s) caused mortality of certain insect life forms and
disruption of the reproductive capacity of others in the
population.
[0008] An advantage of the invention is to provide novel and highly
effective compositions and methods for controlling social insect
populations.
[0009] It is an advantage of the invention to provide
cost-effective and biologically-based natural compounds to control
target populations of fire ants.
[0010] It is another advantage of the present invention to provide
a novel use of an insect neuromodulator and/or derivative(s)
thereof for controlling target insect populations which has
heretofore not been known to have deleterious effects on insect
populations.
[0011] An additional advantage of the invention is to provide novel
methods of controlling target ant populations with a reduced or
eliminated need for potentially harmful and dangerous conventional
chemical control methods.
[0012] It is also an advantage of the current invention to provide
methods of controlling target ant populations with compositions
that have no known effects on vertebrates.
[0013] A further advantage of the invention is to provide methods
of controlling target ant populations with compositions that have
specificity for particular ant species.
[0014] It is a further advantage of the present invention to
provide safe and effective methods of controlling insect
populations while maintaining ecological integrity.
[0015] It is yet another advantage of the present invention to
provide environmentally safe and efficacious methods of controlling
and/or eliminating fire ant populations using dosages of a
conveniently available naturally-occurring ant neuromodulator
and/or its derivatives.
[0016] Another advantage of this invention is to provide novel
biologically-based methods to manage invasive and native pest ant
populations in urban and rural environments.
[0017] This summary is provided to introduce a selection of
concepts in a simplified form that are further described below in
the detailed description. This summary is not intended to identify
all key or essential features of the claimed subject matter, nor is
it intended as an aid in determining the scope of the claimed
subject matter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1A-1C illustrate quantitative results for tyramine in
mature female alate (MFA) and newly mated queen (NMQ) heads,
thoraces, and abdomens by HPLC-MS, measured as detector response
units.
[0019] FIG. 2A-2B shows data for establishing the enzymatic
hydrolysis of acetyl and hexanoyl tyramide via enzymes found in the
MFA reproductive system.
[0020] FIG. 3A-3B shows the percent loss of acetyl and hexanoyl
tyramide over time when incubated with tyramide hydrolase.
[0021] FIG. 4A-4B shows hydrolysis and temperature range for
efficient conversion of acetyl and hexanoyl tyramides to tyramine,
which corresponds to fire ant mating flight temperatures.
[0022] FIG. 5A-5B show phenotypic effects of tyramine and acetyl
tyramide on MFAs after injection, including an illustration that
injection of tyramine (5A) or acetyl tyramide (5B) causes faster
and inappropriate dealation in MFAs, when compared to the
control.
[0023] FIG. 6A-6B show the effect of water control, tyramine,
acetyl tyramide, and hexanoyl tyramide on ovariole development when
injected into MFAs.
[0024] FIG. 7 shows accumulated worker mortality after feeding on a
sucrose solution containing tyramine or tyramides.
[0025] FIG. 8A-8B shows worker mortality after feeding on a sucrose
solution containing tyramine or acetyl tyramide in fully functional
sub-colonies.
[0026] FIG. 9A-9B shows worker mortality for three concentrations
of tyramine fed as droplets to worker groups.
[0027] FIG. 10 shows the effects of tyramine and acetyl tyramide
hand fed to larvae.
[0028] FIG. 11A-11B illustrate that (A) feeding tyramine and/or the
tyramides to colony sub-units that contained immature female alates
results in inappropriate wing loss, as well as ovariole development
(data not shown), and (B) worker mortality, and likely wing muscle
histolysis and queen pheromone production.
[0029] FIG. 12A-12B shows mortality results for five biogenic
amines fed to fire ant workers.
[0030] FIG. 13 shows that tyramine has specificity for certain ant
species and does not cause worker mortality in all ant species.
[0031] FIG. 14 shows fire ant worker mortality was significantly
different from a control when fed tyramine at a 100-fold range of
concentrations.
[0032] FIG. 15 shows fire ant worker mortality was significantly
different from a control when fed N-methyl-tyramine over a 100-fold
range of concentrations.
[0033] FIG. 16 shows fire ant worker mortality was significantly
different from a control when fed a 100-fold range of
concentrations of N-methyl-tyramine (HCl).
[0034] FIG. 17 shows a comparison of the effects of like
concentrations of tyramine and N-methyl tyramine (HCl) versus a
control, after being fed to fire ant workers.
[0035] FIG. 18 shows a comparison of the effects of like
concentrations of tyramine and N-methyl tyramine (HCl) versus a
control, after being fed to fire ant workers.
[0036] FIG. 19 shows a comparison of the effects of like
concentrations of tyramine and N-methyl tyramine (HCl) versus a
control, after being fed to fire ant workers.
[0037] FIG. 20 shows fire ant mortality results for two
concentrations of 3-chloro-4-methoxyphenethylamine versus a
control, after being fed to fire ant workers.
[0038] FIG. 21 shows worker mortality results for tyramine
derivatives when the 4-hydroxyl group is replaced by a halogen:
4-chloro-, 4-bromo-, and 4-flurophenethylamine, after being fed to
fire ant workers.
[0039] FIG. 22 shows a comparison of planar ovariole area (pixels)
measurements of treatment and control sub-colonies composed of
female sexuals with workers, but no queen, after being fed the
indicated treatments/control, and after the female sexuals lost
their wings.
[0040] FIG. 23 shows results from a bioassay that measured
attraction of workers to female sexual poison gland extracts, after
the female sexuals lost their wings.
[0041] FIG. 24A-24B show mortality was significantly different from
a control when mongyne queenright colonies were fed tyramine or
N-methyl-tyramine. FIG. 24A shows worker mortality and FIG. 24B
shows queen mortality.
DETAILED DESCRIPTION OF THE INVENTION
[0042] Unless herein defined otherwise, all technical and
scientific terms used herein generally have the same meaning as
commonly understood by one of ordinary skill in the art to which
this invention belongs. Any definitions below may or may not be
used in capitalized form herein and are intended to be used as a
guide for one of ordinary skill in the art to make and use the
invention and are not intended to limit the scope of the invention.
Mention of trade names or commercial products is solely for the
purpose of providing specific information or examples and does not
imply recommendation or endorsement of such products.
[0043] The term "consisting essentially of" excludes additional
method (or process) steps or composition components that
substantially interfere with the intended activity of the method
(or process) or composition. This term may be substituted for
inclusive terms such as "comprising" or "including" to more
narrowly define any of the disclosed embodiments or
combinations/sub-combinations thereof. Furthermore, the exclusive
term "consisting" is also understood to be substitutable for these
inclusive terms.
[0044] The term "effective amount" of a compound or property as
provided herein is meant such amount as is capable of performing
the function of the compound or property for which an effective
amount is expressed. As is pointed out herein, the exact amount
required will vary from process to process, depending on recognized
variables such as the compounds employed and various internal and
external conditions observed as would be interpreted by one of
ordinary skill in the art. Thus, it is not possible to specify an
exact "effective amount," though preferred ranges have been
provided herein. An appropriate effective amount may be determined,
however, by one of ordinary skill in the art using only routine
experimentation.
[0045] The term "optional" or "optionally" means that the
subsequently described event or circumstance may or may not occur,
and that the description includes instances in which said event or
circumstance occurs and instances where it does not. For example,
the phrase "optionally comprising a defoaming agent" means that the
composition may or may not contain a defoaming agent and that this
description includes compositions that contain and do not contain a
foaming agent.
[0046] The term "substantially pure" refers to a formulation that
is at least about 90% (e.g., at least 90%) in purity weight/weight
of a total composition. In a more preferred embodiment, the purity
is at least about 95% (e.g., at least about 95%) weight-to-weight,
or at least about 98% (e.g., at least about 98%) purity.
[0047] In invertebrates, the biogenic amines octopamine (OA) and
tyramine (TA) are functional homologs to the vertebrate adrenergic
neurotransmitters, noradrenaline and adrenaline. They are the only
known non-peptide neurotransmitters/hormones found in insects and,
interestingly, these biogenic amines have no known activity in
vertebrates (see Roeder, T. (2005), Tyramine and octopamine: ruling
behavior and metabolism. Ann. Rev. Entomol. 50:447-477) Roeder,
Ann. Rev. Entomol. 50:447-477). Biogenic amines are normally
biosynthesized and stored in the insect brain. When released into
the hemolymph they bind to corresponding G-Protein-coupled
receptors (GPCRs) (e.g., tyramine receptor (TAR) and octopamine
receptor (OAR)) and elicit a response. As a consequence of the OA
and TA invertebrate specificity, there has been research to use the
system for insect control through identification of agonists (e.g.,
to mimic receptor activation) and/or antagonists (e.g., to block
the action of the receptor) of the OAR and, to a lesser extent,
TAR. TA is the natural ligand for TAR and until the disclosure of
this invention, has not been considered to have negative effects on
insects.
[0048] As described herein, tyramine is normally a neuromodulatory
biogenic amines that have been shown to have critical developmental
and reproductive functions in insects. Related compounds,
tyramides, are found only in males of the ant species in the
Myrmicinae sub-family. Tyramides are transferred into the female
sexual during mating and are, in turn, quickly hydrolyzed to
tyramine (see FIGS. 1A-1C & 2A-2B). Tyramine floods the
hemolymph of the now newly mated queen (NMQ) to act on tyramine
receptors to trigger wing loss, ovariole development, and other
physiological changes in the NMQ quickly after mating. There is no
rational linkage between the normal endocrinology of tyramine and
the present invention. The discovery that certain dosages of
tyramine act to control and/or eliminate populations of fire ants
(e.g., killing worker ants) was entirely surprising and unexpected.
Also surprising is that tyramine does not have the same impact on
all ant species. For example, tawny crazy ant (Nylanderia fulva)
males do not produce tyramides, but they need to accomplish an
analogous result after mating (data not shown). Similar testing
with tyramides and tyramine for tawny crazy ant mortality revealed
no effect on crazy ant workers (see example 13 below).
Additionally, testing fire ants with tyramine and other biogenic
amines that may be found in insects (e.g., dopamine, histamine,
octopamine, serotonin), surprisingly revealed the desired results
specifically with the delivery of tyramine, while the other
biogenic amines did not result in significant mortality (see
example 12 below). It was equally surprising that tyramine fed to
fire ant MFAs by workers in sub-colonies resulted in rapid wing
loss and commencement of ovariole development bypassing the normal
route of mating flight and mating. In addition, until now ovariole
development in MFAs was thought to be held in check by colony queen
primer pheromones by inhibition of MFA Juvenile Hormone (JH)
release (see Vargo, E. L. (1998), Primer pheromones in ants, pp.
293-313. In R. K. Vander Meer, M. D. Breed, K. E. Espelie and M. L.
Winston (eds.), Pheromone communication in social insects ants,
wasps, bees, and termites. Westview Press, Boulder, Colo.).
[0049] In an embodiment, only tyramine is used in the disclosed
methods as the delivered tyramine composition. In other
embodiments, tyramine is combined with one or more of its
derivatives as the delivered tyramine composition. In yet other
embodiments, only one or more tyramine derivatives are used as the
delivered tyramine composition. As used herein the term "tyramine"
is meant to be inclusive or exclusive according to the context of
only (i) tyramine, (ii) tyramine and at least one of its
derivatives, or (iii) at least one of its derivatives without the
presence of tyramine. For example, tyramine in the absence of its
derivatives means the delivery of only tyramine as active
ingredient and tyramine and at least one of its derivatives means
the delivery of tyramine and one or more of its derivatives as
active ingredient. It should be appreciated that any such single
active ingredient or combinations of active ingredients may be
delivered via any of the mechanisms herein described. Not all
tyramine derivatives may exhibit the desired activity and this
invention is contemplated to selectively include any one or more of
those derivatives of tyramine which exhibit the desired activity or
elicit the desired response. There are a number of commercially
available tyramine derivatives that may have enhanced activity
against fire ants or other pest ants. Among those are, for example,
N-(4-chlorobenzoyl) tyramine; N-benzoyl-tyramine; N-formyltyramine;
4-acetlyltyramine; tyramine O-sulfate; 3-fluoro-tyramine;
methyl-4-tyramine; 2-tyramine; m-tyramine; 3,5-diiodo-tyramine;
N,N-di-n-propyl-m-tyramine; 3-methyl-tyramine;
N,N-dimethyl-tyramine; the like; and any combinations thereof.
Screening compounds such as these can be carried out by methods
already developed for tyramine itself as shown in the examples
below.
[0050] In an exemplary embodiment, tyramine and/or its derivatives
are delivered in a solution or formulation (e.g., sucrose or other
formulation attractive to and ingestible by the desired ant
population) as an ingestible bait formulation. Sucrose is a food
source that induces the ant to ingest it upon tasting it. A
substance that induces this type of behavioral action is generally
called a phagostimulant. Other examples of fire ant phagostimulants
include soybean or corn oil which are used as the active ingredient
solvent and phagostimulant. In the present invention, tyramine is
water soluble, so the oils may not function as desired unless other
additives are used to create an emulsion or make the tyramine
miscible or dispersible in the oil. Such methods are known in the
art and the particular formulation may be selected by one having
skill in the art. In this way, the formulation exerts two major
effects: (i) workers and larvae exhibit significant mortality and
(ii) female alates in experimental colonies inappropriately exhibit
wing loss and begin ovariole development, laying uninseminated eggs
which develop into males, and also may produce queen pheromone
while in their mother colony. The negative effects of significant
mortality are obvious and severely detrimental to colony health.
For (ii) the negative effects are two-fold. First, the female
alates begin mimicking queens in direct competition with the real
queen for colony resources and the eggs they lay are infertile
resulting in non-functional males, and second, increased levels of
queen pheromone results in lower fecundity of the real colony queen
(i.e., colony size decreases).
[0051] Not wishing to be bound to a particular theory, the
compounds described herein (i.e., tyramine and its derivatives)
provide control for ant populations by, for example, disrupting the
availability of potential queens, thereby reducing fire ant
reproductive potential. In some embodiments, the compositions are
used (e.g., applied or devices are deployed) in or prior to the
spring season when colonies are producing male and female alates
for mating flights. The compounds described herein can also be
incorporated into compositions and devices useful in controlling
insect pests (e.g., ants such as ants of the subfamily Myrmicinae).
For example, the compounds described herein can be incorporated
into a composition for application to the environment of the pest,
optionally with an agriculturally suitable carrier comprising at
least one of a liquid diluent, a solid diluent, or surfactant as
further described herein.
[0052] In an embodiment, the invention is a method of controlling a
population of insects (e.g., a colony or a plurality of colonies of
insects including social insects). While it should be appreciated
that the method is applicable to a variety of different insects
including social insects, the target insect is preferably ants,
and, in particular, the method relates to controlling populations
of ant species having a functional sting, such as those in the
genus Solenopsis, one of which is S. invicta, known as the "red
imported fire ant," and those in the Myrmica genus, one of which is
Myrmica rubra, also known as the European fire ant or common red
ant. In embodiments, the compounds, compositions, and formulations
described herein can be used in methods for controlling ants, for
example, ants of the subfamily Myrmicinae. For example, the methods
can be used to control pest species in the following genera:
Monomorium, Myrmicaria, Solenopsis, Megalomyrmex, Trachymyrmex,
Cyphomyrmex, Tetramorium, Pheidole, Myrmica, Crematogaster,
Pogonomyrmex, and Atta.
[0053] The method includes delivering an effective amount of a
tryamine composition (the structure of tyramine is shown below) to
the population of insects. It should be appreciated, as discussed
herein, that the tyramine composition may include tyramine and/or
its derivatives as the active ingredient.
##STR00001##
[0054] Tyramine and its derivatives are commercially available from
a variety of sources. For example, LabNetwork (www.labnetwork.com),
eBiochem (www.ebiochem.com), and Cambridge Chemicals (Boston,
Mass.) have tyramine available. In addition, methods of
synthesizing tyramine and its derivatives are known in the art (see
e.g., Shimizu, Y., et al., (2012), Microwave-Assisted Deacylation
of Unactivated Amides Using Ammonium-Salt-Accelerated
Transamidation. Angew. Chem. Int. Ed., 51: 8564-8567; Zhang, H., et
al., (2016), Two-step enzymatic synthesis of tyramine from raw
pyruvate fermentation broth. J. Mol. Catal. B: Enzym., 124: 38-44;
Santaniello, E., et al., (1981), On the stereochemistry of the
decarboxylation of (2S)-histidine catalysed by histidine
decarboxylase from Clostridium welchii (E.C.4.1.1.22). J. Chem.
Soc., Perkin Trans. 1, 307-309; Rastetter, W. H. and Nummy, L. J.
(1980), epidithiadiketopiperazines. Biogenesis of Synthesis of the
three isomeric (.beta.-aminoethyl)benzene oxides. J. Org. Chem.,
45(16): 3149-3155).
[0055] In embodiments, the invention is a composition including an
effective amount of a compound selected from the group consisting
of: tyramine, one or more derivatives of tyramine, and combinations
thereof. The effective amount is sufficient to control a target
population of social insects and is described herein in relation to
the treatment methods for the target population(s) of social
insects. The composition may include one or more various liquid
diluents, solid diluents, surfactants, additives, preservatives,
and combinations thereof as described herein. The composition may
be in the form of a substantially pure composition, or, in other
embodiments as a solid, liquid, semi-solid, spay, powder, granule,
tablet, gel, cream, lotion, the like, and combinations thereof as
further described below.
[0056] The effective amount of tyramine delivered to the population
may be calculated as a multiple of the amount on a per insect basis
(e.g., worker ant, fire ant worker) of an average amount of
naturally-occurring tyramine in individual insects, such as within
the brains or other parts of individual insects (e.g., worker ant,
fire ant worker). For example, it is estimated that "normal" fire
ant workers have an amount of tyramine in their brains ranging from
about 250 picograms to about 600 picograms (e.g., individual fire
ant worker brains contain about 250 pg of octopamine on average and
about 600 pg of dopamine on average (see Vander Meer, R. K., et
al., (2008), Queen regulates biogenic amine level and nestmate
recognition in workers of the fire ant, Solenopsis invicta.
Naturwissenschaften 95(12): 1155-1158). Fire ant sexual larvae die
when fed 0.8 micrograms tyramine and it is estimated from feeding
studies (see examples below) that 0.01 micrograms per microliter
solution of tyramine fed to workers led to mortality after 7 days.
These values were empirically obtained and are much greater than
the naturally occurring amount. An effective amount of tyramine
delivered to an insect population is, therefore, preferably from
about 0.01 microgram to about 2.0 micrograms (e.g., 0.01 microgram
to 2.0 micrograms) of tyramine active ingredient per fire ant
worker, according to an embodiment. A preferred range of tyramine
active ingredient is from about 0.1 micrograms to about 2
micrograms (e.g., 0.1 microgram to 2 micrograms), or from about 0.2
microgram to about 3 micrograms (e.g., 0.2 microgram to 3
micrograms), or from about 0.3 microgram to about 4 micrograms (0.3
microgram to 4 micrograms) per fire ant worker. It should be
appreciated that much higher amounts of tyramine and/or its
derivatives could also be delivered to the insect population (e.g.,
2 to 5 micrograms, 5 to 10 micrograms, 10 to 20 micrograms per fire
ant worker, or higher).
[0057] In an embodiment, the amount of tyramine effective to
produce the surprising and unexpected results of the invention may
also be calculated as a multiple of the amount of tyramine present
in the brain of an individual insect (e.g., ant, worker ant, fire
ant worker). In a preferred embodiment, the amount of total
tyramine delivered to a population is equivalent to at least about
1,000,000 (e.g., 1,000,000) times the amount of tyramine present on
average on a per insect basis within the population, or at least
about 100,000 (e.g., 100,000) times, or at least about 50,000
(e.g., 50,000) times, or at least about 25,000 (e.g., 25,000)
times, or at least about 10,000 (e.g., 10,000) times, or at least
about 1,000 (e.g., 1,000) times, or at least about 500 (e.g., 500)
times, or at least about 100 (e.g., 100) times, or at least about
50 (e.g., 50) times, or from about 50 to about 1,000 times (e.g.,
50 to 1,000), or any value within such ranges as selected by a
skilled artisan. For example, if an individual fire ant worker has
from about 250 picograms to about 600 picograms of tyramine in its
brain and there are 50,000 fire ant workers in the population, the
amount of tyramine active ingredient delivered would be at least
about 0.05 grams. For another example, 1.25 grams could be used for
50,000 fire ant workers to deliver about 25 micrograms per worker
(or about 25,000,000 picograms per worker). This delivered amount
is about forty two thousand times the 600 picogram maximum value
normally found in a worker ant brain. If one microgram was needed
per worker for a particular target population, 50,000 workers would
ingest a total of about 50 mg of tyramine or 0.05 g.
[0058] The amount of active ingredient in the compositions of the
invention is/are present in an amount sufficient to deliver the
prescribed dosages to the insect populations based on, for example,
an amount to be delivered to a target insect population on a per
insect basis and/or a concentration range of the active ingredient.
A preferred concentration for the active ingredient of tyramine
and/or derivatives thereof is at least about 0.0001% on a weight
per volume basis, or a range from about 0.0001% (e.g., 1,000
pg/.mu.l) to about 1% (e.g., 1 mg/ml) on a weight per volume basis,
or from about 0.001% to about 1%, or from about 0.01% to about 1%.
Higher concentrations may also be used but are generally not usable
due to the solubility limitations of tyramine and derivatives
thereof in aqueous solutions. The composition may also be prepared
from a more concentrated form such as a solid or powder. For
example, the active ingredient may be mixed with a solid diluent or
in combination with a phagostimulant which the user would mix with
an aqueous diluent to produce a phagostimulant. In another example,
tyramine and/or its derivatives could be pre-formulated with the
appropriate amount of phagostimulant (e.g., sucrose or other
phagostimulants selected by a skilled artisan for a particular
application) in a sachet or other container that would preserve its
integrity until use for pest ant control. The sachet could be
opened and poured into the correct pre-measured amount of an
aqueous diluent. Alternatively, the sachet type is selected from a
variety of materials, such as biodegradable or bio-based plastic.
In another example, the sachet is water-soluble plastic which could
dropped into a pre-measured amount of water to achieve an aqueous
composition having the desired concentration. The solution would
then be used to charge a bait station dispenser (discussed below).
The bait station dispenser could be charged as needed with a
freshly prepared composition as often as needed. As further
discussed herein, another embodiment of the solid ingredients is
tyramine combined with phagostimulant and optional preservative.
Compatibility of such ingredients would be determined for a given
application by a skilled artisan. In another embodiment, the
aqueous formulation comprises tyramine and/or its derivative
combined with a phagostimulant and an optional preservative which
is then absorbed into an inert material or matrix from which the
target insect could access the liquid from the inert material. In a
further embodiment, the liquid formulation could be encapsulated
(e.g., hydrogels or other such material as discussed below). The
sachet or encapsulating material containing the composition may be
of such construction that the target insect could sense the
phagostimulant and break open the encapsulation to access the
composition within the capsule or sachet.
[0059] The formulation or composition ingredients are selected to
be consistent with the physical properties of the active
ingredient, mode of application (e.g., bait design or bait station
type) and environmental factors such as soil type, moisture, and
temperature. Useful formulations include liquids such as solutions
(e.g., including emulsifiable concentrates), suspensions, emulsions
(e.g., including microemulsions and/or suspoemulsions) and the like
which optionally can be thickened into gels. Useful formulations
further include solids such as dusts, powders, granules, pellets,
tablets, films, and the like which can be water-dispersible (e.g.,
wettable) or water-soluble. Active ingredient can be encapsulated
or microencapsulated and further formed into a suspension or solid
formulation; alternatively the entire formulation of active
ingredient can be encapsulated or microencapsulated (e.g., to
control or delay release of the active ingredient). Sprayable
formulations can be suspended in suitable media and used at
preferred spray volumes (e.g., from about one to several hundred
liters per hectare). High-strength compositions are primarily used
as intermediates (e.g., concentrates) for further formulation
(e.g., by dilution).
[0060] Solid diluents are known in the art (e.g., as described in
Watkins, et al., Handbook of Insecticide Dust Diluents and
Carriers, 2nd Ed., Dorland Books, Caldwell, N.J.). Exemplary solid
diluents can include, for example, clays such as bentonite,
montmorillonite, attapulgite and kaolin, starch, sugar, silica,
talc, diatomaceous earth, urea, calcium carbonate, sodium carbonate
and bicarbonate, and sodium sulfate. Liquid diluents are known in
the art (e.g., as described in Marsden, Solvents Guide, 2nd Ed.,
Interscience, New York, 1950). Exemplary liquid diluents include,
for example, water, N,N-dimethylformamide, dimethyl sulfoxide,
N-alkylpyrrolidone, ethylene glycol, polypropylene glycol,
paraffins, alkylbenzenes, alkylnaphthalenes, oils of olive, castor,
linseed, tong, sesame, corn, peanut, cotton-seed, soybean,
rape-seed and coconut, fatty acid esters, ketones such as
cyclohexanone, 2-heptanone, isophorone and
4-hydroxy-4-methyl-2-pentanone, and alcohols such as methanol,
cyclohexanol, decanol, and tetrahydrofurfuryl alcohol.
[0061] Surfactants are known in the art (e.g., as described in
McCutcheon's Detergents and Emulsifiers Annual, Allured Publ.
Corp., Ridgewood, N.J.; and in Sisely and Wood, Encyclopedia of
Surface Active Agents, Chemical Publ. Co., Inc., New York, 1964).
Surfactants can include, for example, polyethoxylated alcohols,
polyethoxylated alkylphenols, polyethoxylated sorbitan fatty acid
esters, dialkyl sulfosuccinates, alkyl sulfates, alkylbenzene
sulfonates, organosilicones, N,N-dialkyltaurates, lignin
sulfonates, naphthalene sulfonate formaldehyde condensates,
polycarboxylates, and polyoxyethylene/polyoxypropylene block
copolymers.
[0062] In some embodiments, the formulations described herein can
contain minor amounts of additives to reduce foam, caking,
corrosion, microbiological growth and the like, or thickeners to
increase viscosity. Methods for preparing the formulations
described herein are known in the art. For example, solutions,
including emulsifiable concentrates, can be prepared by simply
mixing the ingredients. Dusts and powders can be prepared (e.g., by
blending and, optionally grinding such as using a hammer mill or
fluid-energy mill). Suspensions can be prepared, for example, by
wet-milling (see e.g., U.S. Pat. No. 3,060,084). Granules and
pellets can be prepared by, for example, spraying the active
material upon preformed granular carriers or by agglomeration
techniques (see e.g., Browning, Chem. Eng., p 14748, (1967);
Perry's Chemical Engineer's Handbook, 4th Ed., McGraw-Hill, New
York, 1963, pages 8-57 and following, and PCT Publication WO
91/13546). Pellets can be prepared using methods known in the art
(e.g., as described in U.S. Pat. No. 4,172,714). Water-dispersible
and water-soluble granules can be prepared (e.g., as taught in U.S.
Pat. Nos. 4,144,050, 3,920,442 and DE 3,246,493). Tablets can be
prepared (e.g., as taught in U.S. Pat. Nos. 5,180,587, 5,232,701,
5,208,030, 6,245,816, or 7,696,233; or see also Woods, "The
Formulator's Toolbox--Product Forms for Modern Agriculture" in
Pesticide Chemistry and Bioscience, The Food--Environment
Challenge, T. Brooks and T. R. Roberts, Eds., Proceedings of the
9th International Congress on Pesticide Chemistry, The Royal
Society of Chemistry, Cambridge, 1999, pp. 120-133).
[0063] In embodiments, the composition can be delivered in a solid
(e.g., as pellets or powder formulation); a semi-solid (e.g., a
gel, hydrogel, or gellable formulation), as part of an ant trap; or
in a liquid formulation (e.g., in a spray format), and can be used,
for example, to attract, direct, or repel ants from or to a
selected area. In some embodiments, the compounds described herein
are used as a repellent (e.g., applied to or in an area in which
the presence of ants is not desired). The area can be, for example,
an indoor or outdoor area, in which case the compounds can be
delivered generally (e.g., in a spray, powder, granule, gel,
hydrogel, or other format). In some embodiments, the composition
can be provided in a delayed-release composition (e.g., any method
of dispersal, dispensation, application, timed-release,
encapsulation, microencapsulation, or the like to apply the
composition of the invention as further described herein). In
embodiments, "carriers" may include a variety of
microencapsulation, controlled-release, and other dispersion
technologies available to those of ordinary skill in the art, so
that the composition is released over time. Other suitable
compositions are also known in the art.
[0064] In some embodiments, the compounds described herein can be
formulated with other ingredients (e.g., ant-attractant or
ingredients such as one or more phagostimulants, ant food (e.g.,
carbohydrates, fats, proteins, and/or any other nutritional
components), etc. (see, e.g., U.S. Pat. Nos. 5,152,096; 5,939,061;
6,916,469; and 7,048,918)). Preferred concentrations of sugar in,
for example, an ingestible bait composition is from about 1% to
about 5% (e.g., 1% to 5%), or from about 5% to about 10% (e.g., 5%
to 10%), or from about 10% to about 50% (e.g., 10% to 50%) sugar
weight-to-weight. High concentrations of sugars, for example, have
antimicrobial properties. For example, a 10% sucrose solution as
used in the examples below retarded formation of a color in the
solution for about a week (data not shown). Increasing the
concentration to 50% gave microbial control for about 3 weeks;
however, such high levels of sucrose decrease the amount of
solution ingested. A 1% solution of the disclosed active compounds
(e.g., tyramine or N-methyl tyramine) is at their solubility
maximum. Keeping sucrose levels lower (e.g., about 10%) and adding
a non-nutritive antimicrobial preservative to increase the lifetime
of the solution at ambient temperatures would be beneficial in
alternative embodiments of the invention. Examples of preservatives
are known in the art and may be selected as needed by a skilled
artisan in implementing the invention: citric acid, ascorbic acid,
sodium nitrite, sulfites (e.g. sulfur dioxide), and sodium
erythorbate
[0065] The compounds described herein can be formulated or
emulsified/homogenized, for example, as part of an ant bait to
deliver an ant toxin for extermination of pest insects. A number of
ant toxins are known in the art, including Thiamethoxam (Syngenta
Crop Protection, Greensboro, N.C.); DOT; growth regulators;
orthoboric acid; borates (e.g., sodium tetraborate); abamectin BI
(Whitmire Micro-Gen Research Laboratories, Inc.); fipronil
(2-Phenethyl Propionate); Sulfluramid; Imidacloprid (Bayer
Environmental Science, Montvale, N.J.); indoxacarb (Dupont,
Wilmington, Del.); O,S-Dimethyl Acetylphosphoramidothioate;
2-phenethyl propionate; piperonyl butoxide; pyrethrins (e.g.,
permethrin, deltamethrin, tetramethrin, or Lambda-cyhalothrin
[1.alpha.(S*),3.alpha.(Z)]-(.+-.)-cyano-(3-phenoxyphenyl)methyl-3-(2-chlo-
-ro-3,3,3-trifluoro-1-propenyl)-2,2-dimethylcyclopropanecarboxylate);
or Cry toxin (U.S. Pat. No. 6,797,490). The toxin can be delivered
with the compounds described herein (e.g., as part of a single
composition) or can be delivered before or after delivery of the
compounds described herein. In embodiments, the disclosed tyramine
composition may be delivered as part of a solution such as a sugar
solution (e.g., sucrose, glucose, fructose, and the like). Sucrose
is a very good fire ant worker phagostimulant and also has
antimicrobial activity, exerted via high osmotic pressure. The
higher the concentration the greater the osmotic pressure and the
better the antimicrobial activity.
[0066] In some embodiments, the compounds described herein are used
with an insect trap (e.g., to attract and trap insects) for pest
control or to detect new or ongoing infestations. The traps can
optionally include means for preventing the insect from escaping
the trap. For example, mechanical traps are generally designed to
physically prevent the insect from leaving, such as a pattern that
only allows ingress but not egress, or an edge or flange that
prevents escape. Chemical traps having, for example, an adhesive or
liquid that traps the insect, may also be used. Other examples are
described in U.S. Pat. Nos. 6,966,145, 6,609,330, 6,796,082,
6,283,064, 6,655,079, 6,474,015, 5,746,021, and 5325626. Other
suitable traps are also known in the art.
[0067] In general the present formulations and compounds will be
delivered in an amount effective for the intended purpose for
insect control and can be delivered directly onto an area known or
evident of infestation (e.g., targeted directly onto an ant colony,
or near an area known or suspected of ant infestation). In
embodiments, the disclosed compositions are delivered onto an
agricultural (e.g., hundreds or thousands of acres), residential
(e.g., over less than acre or up to tens of acres), or commercial
area in which ant control is desirable. Various modes of delivery
may be used as herein described. For example, bait stations are
known in the art and a skilled artisan would select an appropriate
bait station or bait design for any set of circumstances where the
invention is to be deployed and used (see e.g., U.S. Pat. Nos.
5,939,061; 5,746,021; 7,941,777). Furthermore, the invention also
provides kits which are useful for carrying out methods of the
present invention. The kit includes a container comprising
compositions of the present invention and instructions for using
the compositions for the purpose of controlling insect populations
as disclosed herein. The kits can comprise a first container means
containing the compositions described herein. The kit can also
comprise other container means having one or more solutions,
diluents, or applicators necessary or convenient for carrying out
the invention. The container means can be made of glass, plastic,
foil, the like, and combinations thereof and can be any suitable
vial, bottle, pouch, tube, bag, box, etc. The kit can also contain
written information, such as procedures for carrying out the
present invention or analytical information, such as the amount of
reagent contained in the first container means. The container means
can be in another container means (e.g., a box, bag, etc.) along
with the written information.
[0068] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical values, however,
inherently contain certain errors necessarily resulting from error
found in their respective measurement. The following examples are
intended only to further illustrate the invention and are not
intended in any way to limit the scope of the invention as defined
by the claims.
EXAMPLE 1
[0069] Analysis of mature female alate (MFA) and newly mated queen
(NMQ) heads, thoraces, and abdomens by HPLC-MS for tyramine yielded
the quantitative results shown in FIG. 1. The Y-axis Units are
detector response. The amount of tyramine within the three graphs
are comparable via the mean detector response value. Note that the
Y-axis scale is different in the three graphs and that the amount
of tyramine in NMQ abdomens is about 20 to 30 times the amount in
NMQ heads and thoraces. The amount of tyramine in NMQs is
significantly greater than the amount found in MFAs for all body
parts. The time from an MFA to NMQ during the mating flight is only
about 60 min and the NMQs collected for this analysis were
refrigerated or frozen within 30 min of collection. Previous
research demonstrated that males store microgram quantities of
acetyl and hexanoyl tyramides in their external genetalia, and that
they transfer these compounds to MFAs during the mating process
(data not shown). These two pieces of information led to the
hypothesis that MFAs have enzymes in their reproductive system that
hydrolyze tyramides to tyramine, which then passes into the now
NMQ's hemolymph to act on tyramine receptors.
EXAMPLE 2
[0070] To test the enzymatic hydrolysis hypothesis discussed in
Example 1, the tip of the MFA reproductive system was dissected,
sonicated in water, then a known amount of synthetic tyramide was
added and incubated at a single temperature (see FIG. 2A-2B) for 60
min. Aliquots were removed at 5 min intervals and added to methanol
to quench the enzymatic reaction, then analyzed for tyramide by
GC/MS. As shown in FIG. 2A-2B, temperatures from 20.degree. C. to
42.degree. C. defined the temperature dependence of the enzyme. At
the end of these experiments, the remaining extract was analyzed
for tyramine (HFBAA derivatization--see Vander Meer, R. K., et al.,
(2008), Queen regulates biogenic amine level and nestmate
recognition in workers of the fire ant, Solenopsis invicta.
Naturwissenschaften 95(12): 1155-1158)) and Expression CMS (Compact
Mass Spectrometer) (Advion Biosciences Inc., Ithaca, N.Y.). The
results confirmed that the tyramides were being converted to
tyramine. To determine the specificity of this enzyme MFA mid- and
hind-gut were dissected, sonicated in water, acetyl or hexanoyl
tyramide was added, and incubated at 32.degree. C. Samples were
taken periodically as described above (data not shown). No
degradation of the tyramide was observed, therefore, the tyramide
hydrolase is specific to the MFA reproductive system.
EXAMPLE 3
[0071] The graphs in FIGS. 3A and 3B were converted to percent loss
to normalize differences in initial tyramide concentration, then
subjected to linear regression to provide the slopes (i.e., rate of
loss) for each incubation temperature. FIGS. 3A and 3B show the
percent loss of acetyl and hexanoyl tyramide over time (shown in
min). It is clear that the tyramide hydrolase has a window of
activity between 20.degree. C. and 40.degree. C. for the acetyl
tyramide and between 24.degree. C. and 40.degree. C. for hexanoyl
tyramide. The male fire ant transfers both tyramides to the female,
but upon hydrolysis both tyramides are converted to tyramine. These
temperature ranges closely correspond to mating flight temperatures
reported in the literature (see e.g., Tschinkel W R. The fire ants.
Cambridge, Mass.: Harvard University Press; 2006. p. 747),
suggesting close evolutionary coupling of the enzyme and mating
flight activity.
EXAMPLE 4
[0072] The rate of loss illustrated in FIG. 3A-3B provides the data
necessary to calculate (under the experimental conditions indicated
in Example 3) the time it would take the NMQ to hydrolyze the mean
amount of the two tyramides in male external genitalia (FIG. 4A=3.0
microgram and FIG. 4B=3.5 microgram), previously determined
(unpublished data, Vander Meer, 2015). The graphs clearly show very
rapid hydrolysis and a clear temperature range for most efficient
conversion of the tyramides to tyramine. The actual rate of
conversion may be modulated by other compounds or sperm also being
transferred to the MFA during mating.
EXAMPLE 5
[0073] While the MFAs are in their mother colony, the queen
releases primer pheromones that inhibit wing loss (sometimes
referred to herein as "dealation"), development of ovaries, wing
muscle histolysis, and pheromone production. This release prevents
reproductive/resource competition between the MFAs and the mother
queen. If MFAs are removed from the colony, they become
disinhibited and eventually (e.g., within days) lose their wings
and start the physiological processes (e.g., wing loss and ovariole
development). MFA also become disinhibited when they leave the
colony on a mating flight; however, in this case the now NMQs lose
their wings within minutes of landing and initiate the above
mentioned physiological/ biochemical changes (e.g., wing loss and
ovariole development). We know from previous experiments that
mating is the trigger, not mating flight initiation or the act of
flying (Burns, S. N., et al. (2002) Identification and action of
juvenile hormone III from sexually mature alate females of the red
imported fire ant, Solenopsis invicta. Journal of Insect Physiology
48: 357-365; Burns, S. N., et al. (2005), The effect of age and
social environment on dealation in Solenopsis invicta (Hymenoptera:
Formicidae) female alates. Florida Entomologist 88(4): 452-457;
Burns, S. N., et al. (2007), Mating flight activity as dealation
factors for fire ant, Solenopsis invicta, female alates. Annals of
the Entomological Society of America 100(2): 257-264). Testing was
performed to determine whether the male-derived tyramides and/or
their NMQ hydrolysis product, tyramine, are the causative agent(s)
that initiate wing loss and the associated physiological changes
that occur in NMQs. FIG. 5A-5B show phenotypic effects of tyramine
and acetyl tyramide on MFAs after injection (100 nL of a 1.0%
saline solution injected into the MFA abdomen using a
nano-injection system, Bioquip Products, Compton Calif.). Wing loss
(dealation) for female alates typically occurs soon after mating.
Injection of tyramine (5A) or acetyl tyramide (5B) causes
significantly faster and inappropriate dealation (not caused by
mating process) in female alates, when compared to the control.
EXAMPLE 6
[0074] FIG. 6A-6B show the effect of water control, tyramine,
acetyl tyramide, and hexanoyl tyramide on ovariole development when
injected into MFAs (100 nL of a 1.0% saline solution (1 microgram)
injected into the MFA (unpublished, Vander Meer 2015) abdomen
(about 50% of what the males have available to transfer to the MFA
are using a nano-injection system, Bioquip Products, Compton
Calif.). Two measures of ovariole development were used. In FIG. 6A
control and treatment ovarioles were compared to a series of 4
ovary-ranking photos where a rank of 1 was the least developed and
a rank of 4 was the highest. It was surprisingly discovered that
injection caused significant ovariole development in all treatments
compared to the control, FIG. 6A. Measurement of ovariole area in
the same samples gave a quantitative measure of ovariole
development. Tyramine and acetyl tyramide, but not hexanoyl
tyramide caused significant ovariole development compared to
controls, FIG. 6B. In addition, injection surprisingly initiated
inappropriate egg-laying by the treated female alates--sometimes
surprisingly coupled with dealation and sometimes independent of
dealation. Within the context of a colony this effect would be in
direct conflict with the colony queen. Eggs laid would not be
fertile and therefore develop into males. Queen pheromone is also
produced by dealates, which would act to reduce the fecundity of
the colony queen. For example, in polygyne colonies the higher the
number of queens the lower is the mean egg-laying rate/queen due to
the presence of excess queen pheromone (see Vander Meer, R. K., et
al. (1992), A comparison of queen oviposition rates from monogyne
and polygyne fire ant, Solenopsis invicta, colonies. Physiological
Entomology 17(4): 384-390).
EXAMPLE 7
[0075] For commercial use of controlling social insect populations,
the compounds must show phenotypic effects through feeding, as is
necessary for bait development. Baits take advantage of the insects
(e.g., fire ants) excellent resource recruitment system that
requires less active ingredient and the user does not have to
locate all colonies if the bait is distributed on the general
foraging surface or distributed in bait stations. Experiments were
designed to detect phenotypic effects induced through feeding
tyramides and tyramine to fire ants. The experimental units were
queenright sub-colonies (i.e., queen (polygyne), brood and
workers). Workers were allowed to feed on a 10% sucrose solution as
control or with approximately 1% (w/v) of the tyramides or
tyramine. FIG. 7 shows accumulated worker mortality at 9 days after
start of experiment. Surprisingly high worker mortality was
observed from each of the three treatments (ns=not significant),
thus the treatments were not significantly different from each
other. However, all treatments were significantly different from
the sucrose controls (t-test, unpaired, parametric, 2-tailed,
P=0.0029). The starting number of workers was about 300, and in
some replicates very few workers were left to tend the queen and
brood.
EXAMPLE 8
[0076] To determine how quickly the tyramine and acetyl tyramide
act, 60 workers were allowed to feed on tyramine (FIG. 8A) or
acetyl tyramide (FIG. 8B) dissolved at about 1% in 10% sucrose
solution. The control and treatments were presented to the worker
ants as droplets on phase separation filter paper. Consumption of
the droplets was readily observed. Mortality was monitored for each
of the first 10 hours and then at 24 hours. As seen in FIGS. 8A and
8B, both treatments had significantly greater worker mortality than
the sucrose control (Kaplan-Meier mortality curve, Log-rank,
Mantel-Cox test). Tyramine and acetyl tyramide had >50% and
>40% mortality, respectively within the 24 h time period of the
experiment. The observed rapid mortality and potential abnormal
behavior before death suggested that the fed workers might not
distribute the ingested sucrose solution to their nestmates as
would normally be the case. This distribution is a cornerstone of
pest ant baits. To test this hypothesis, a similar experiment was
conducted. Workers were allowed to feed on the treatment and
control solutions and were then transferred to another container
with unfed nestmates. In all cases, it was observed that the
treatment fed workers regurgitated the treatment and sucrose
solutions to their unfed nestmates through the classic mechanism of
trophallaxis (data not shown). Thus, normal worker behavior
necessary for bait development was not affected by the treatments
under these experimental conditions.
EXAMPLE 9
[0077] One criteria for an effective pest ant Active Ingredient
(AI) is maintenance of activity over 1-3 orders of magnitude,
because as the bait is distributed from one ant to another the
material is diluted. The AI must maintain activity until all
individuals in the colony (e.g., workers, brood and sexuals
including the queen) have received the AI. FIG. 9A-9B shows three
concentrations of tyramine (about 1.0, 0.1, and 0.01%
(weight-to-volume) fed to worker groups as described above.
Kaplan-Meier mortality curves are shown in FIG. 9A-9B. It was
surprisingly observed that significant worker mortality is observed
even at a dilution factor of 100 (FIG. 9B, Log-rank, Mantel-Cox
test). Similar results were observed for acetyl tyramide and to a
lesser extent hexanoyl tyramide. Delayed AI activity is an
important factor for ant baits. The 1.0 and 0.1% concentrations had
at least a 5 h mortality delay which is likely enough to allow
complete distribution of the bait throughout the colony life forms
(Oi, D. H., et al. (2006), Speed of efficacy and delayed toxicity
characteristics of fast-acting fire ant (Hymenoptera: Formicidae)
baits. Journal of Economic Entomology 99(5): 1739-1748). The 0.01%
had >24 h delay in mortality.
EXAMPLE 10
[0078] To determine the effects of tyramine and the acetyl tyramide
on larvae, a bioassay was developed and used that allowed direct
feeding of 4th instar (i.e., last instar) sexual larvae known
amounts (80 nL max) of test solutions (treatments were at close to
near-maximum soluble concentration (about 1% weight-to-volume) in a
10% sucrose solution (weight-to-volume). Fourth instar sexual
larvae were used because worker 4th instar larvae are too small to
avoid over feeding, which usually results in death. Larvae (N=10)
were fed once, then observed for phenotypic effects (FIG. 10).
Surprisingly, after day one of the tyramine treatment the surviving
larvae appeared sick and started to ooze fluid. All sexual larvae
fed tyramine died by day 5. None of the controls died during this
time period, but surprisingly, none of the acetyl tyramide
treatments died or appeared sick. It was concluded that the 4th
instar larvae are refractory to the mortality effects observed when
workers are fed tyramine.
EXAMPLE 11
[0079] In this example, whether feeding tyramine and/or the
tyramides to colony sub-units that contained female alates would
result in inappropriate wing loss, as well as ovariole development.
Wing muscle histolysis, and queen pheromone production normally
accompanies wing loss (see Vargo, E. L. (1998), Primer pheromones
in ants, pp. 293-313. In R. K. Vander Meer, M. D. Breed, K. E.
Espelie and M. L. Winston (eds.), Pheromone communication in social
insects ants, wasps, bees, and termites. Westview Press, Boulder,
Colo.). Queenless colonies with brood, workers, and low weight
(immature) female alates (n=5, 3 replicates per treatment and
control) were setup. The units were fed tyramine and tyramides in
10% sucrose continuously for 7 days. The results shown in FIG. 11A
surprisingly showed wing loss in all treatments significantly
greater than control. The data supports and confirms that workers
are feeding the treatments to the female alates, which in turn
stimulates wing loss in the alates. FIG. 11B is from the same
experiment confirming the worker mortality effects of the tyramine
and tyramide treatments.
EXAMPLE 12
[0080] Tyramine is one of five biogenic amines that may be found in
insects (e.g., dopamine, histamine, octopamine, serotonin, and
tyramine). The experiment in this example tests whether fire ant
worker mortality effects observed with tyramine may be a general
result of the ants ingesting greater than normal amounts of a
biogenic amine. To test this hypothesis groups of 20 fire ant
worker ants were starved for 24 hours and fed a 1% (w/v) biogenic
amine in 10% sucrose solution. Treatments and sucrose were
presented to the worker ants as a 25 microliter droplet on a 2
cm.sup.2 piece of silicon treated filter paper. Each treatment and
control were performed in triplicate for a total of 60 worker ants
per treatment and control. Mortality was monitored for the first
7-8 h after introduction of the sucrose solutions, then at 24 h.
The feeding/monitoring sequence was repeated for another 24 h
period for a total of 48 h. The results are shown in FIG. 12A-12B
in the form of Kaplan-Meier mortality curves. FIG. 12A shows
results for five biogenic amines at 1% as their HCl salts. It is
quite surprising and clear that tyramine HCl resulted in
statistically greater mortality than the control and the other 4
biogenic amines. Thus, the effect of tyramine is surprisingly not a
general biogenic amine effect and is specific to tyramine. FIG. 12B
illustrates that results for the tyramine HCl and the tyramine free
base are not significantly different.
EXAMPLE 13
[0081] To evaluate the effect of tyramine on another pest ant
species triplicate groups of 20 Tawny crazy ant (Nylanderia fulva)
workers and triplicate groups of 20 fire ant workers were setup.
Corresponding controls for both pest ant species were workers fed
10% sucrose solution. The treatment was 1% tyramine in 10% sucrose
(weight-to-volume). The control and treatments were presented to
the worker ants as droplets on phase separation filter paper.
Consumption of the droplets was readily observed. Mortality was
monitored for each of the first 10 hours and then at 24 hours. The
results are shown as mean.+-.standard error of accumulated
mortality for controls and treatments at 24 h in FIG. 13.
Surprisingly, the tawny crazy did not have significant mortality
compared to its sucrose control; however, as observed previously,
fire ant workers had significant mortality compared to its sucrose
control. These unexpected results illustrate that the toxicity
effects of tyramine on fire ants are not automatically extended to
other ant species.
EXAMPLE 14
[0082] This Example relates to FIG. 14 to 23. Unless otherwise
noted, the experiments were conducted using the following
protocols.
[0083] Source of Fire Ant Workers.
[0084] Solenopsis invicta colony fragments were collected by
excavation from field populations near Gainesville, Fla., known to
have the monogyne (single queen) social form, based on aggression
bioassays and genetic analysis (see e.g., Morel L, et al.,
Comparison of nestmate recognition between monogyne and polygyne
populations of Solenopsis invicta (Hymenoptera: Formicidae). Annals
of the Entomological Society of America. 1990; 83(3):642-7); Valles
S M & Porter S D. Identification of polygyne and monogyne fire
ant colonies (Solenopsis invicta) by Multiplex PCR of Gp-9 alleles.
[poster paper]. p. 116, In: L. Greenberg & C. Lerner [ed.],
Proceedings of the 2003 Red Imported Fire Ant Conference, Mar.
30-Apr. 1, 2003, Palm Springs, Calif., 145 p.; 2003). For these
experiments, colony fragments containing workers, brood, sometimes
male and female sexuals were quickly shoveled into a 19 L bucket
with its upper inside surface dusted with talcum powder to prevent
escapes. In the laboratory, colony life forms were removed from the
soil by slowly flooding the containers as previously described (see
e.g., Banks W A, et al., Techniques for collecting, rearing, and
handling imported fire ants. USDA, SEA, AATS-S-21, 9 p.; 1981) and
placed in colony trays. All colonies with or without a queen were
provided crickets, water, and 10% sucrose solution and maintained
in the laboratory under ambient conditions. These colonies tested
negative for SINV-3 virus, which if present could confound
interpretation of the results. Female sexuals were collected from
fully functional queenright colonies (i.e., colonies containing
queen, brood, workers, and sexuals).
[0085] Initial Feeding Experimental Procedure.
[0086] Preliminary experiments were conducted to probe the effects
of test compounds on fire ant workers. Worker fire ants (n=20) were
starved overnight and placed in 120 mL plastic cups with the upper
inner surface coated with Fluon.RTM.. The ants in plastic cups were
placed in a tray (50.times.40.times.12 cm) with its inner sides
coated with Fluon. Three plastic containers (250 mL) were filled
with water and placed in each experimental tray to keep the
relative humidity high. A 2.times.2 cm piece of phase separation
(hydrophobic) filter paper was placed in each cup containing worker
ants. Treatments were dissolved in 10% sucrose solution at 1%
(w/v). The control was 10% sucrose. Droplets (25 .mu.l) of
treatments or controls were deposited by syringe on the phase
separation filter paper in each treatment and control. The number
of ants feeding at the droplet was recorded every 5 min for the
first hour, then every hour for a total of 8 h. If the ants
consumed the droplet within the 8 h of the experiment (about 4 to 5
hr), another 25 .mu.l droplet was provided. Mortality was recorded
each hour and at 21-24 h (total of 12 evaluations).
[0087] Test Tube Feeding Experimental Procedure.
[0088] Worker ants (250 mg, 300-320 individuals) were weighed and
placed in plastic containers (16.times.4.times.10 cm) coated on the
inside wall with Fluon. Each tray had a nest chamber composed of a
glass test tube (15.times.1.5 cm) filled sequentially with 6 cm
water, cotton plug, and 2 cm of Castone.RTM.. Treatments were
dissolved in 10% sucrose solution at 1% or 0.1%, or 0.01% (w/v).
The control was 10% sucrose. Twelve ml of treatments or controls
were presented to the worker ant bioassay units in a 15 ml test
tube, closed with a cotton plug. Worker ant mortality was recorded
at every 24 h. One previously frozen and thawed cricket was
provided after the first 24 h. The cricket was replaced every other
day. In some experiments, low weight or high weight female alates
(alate/workers combinations were from the same colony) were
included in the experimental units along with the workers. In
addition to monitoring worker mortality, alates were monitored for
wing loss (dealation), ovariole development, and queen pheromone
production. These physiological actions are normally observed
directly after mating. Dead ants were counted and removed each day
and pooled for each replicate. Dealates were also removed
immediately and placed in separate nest chambers, described above.
Dealates from a replicate were pooled in the same chamber. Test
tubes containing treatment or control sucrose were refilled or
replaced as needed during the course of experiment. Dealates were
dissected for measurement of ovariole development and their poison
sac was extracted for olfactometer evaluation within 24 h of wing
loss.
[0089] FIG. 14 shows fire ant worker mortality with 1.0, 0.1, and
0.01% concentrations of tyramine (free base) in 10% sucrose are all
significantly different from the 10% sucrose control. The 1.0 and
0.1% tyramine are not significantly different, but they both are
different from the 0.01% tyramine treatment. Significant mortality
over 100-fold dilution, as seen here, is a key characteristic of an
effective fire ant bait insecticide. The 1.0 and 0.1% are not
significantly different (p=0.08), however, both are different from
the 0.01% solution (p<0.0001).
[0090] FIG. 15 shows fire ant worker mortality with 1.0, 0.1, and
0.01% concentrations of the tyramine derivative, N-methyl-tyramine
(free base) in 10% sucrose. All concentrations are significantly
different from the 10% sucrose control. Significant fire ant worker
mortality for 1.0, 0.1, and 0.01% concentrations of
N-methyl-tyramine (100-fold dilution) is a key characteristic of an
effective fire ant bait insecticide. Interestingly, the two lower
concentrations (0.1 and 0.01%) show significantly higher mortality
than the 1.0% concentration. It is possible that the 1.0% N-methyl
tyramine is not as palatable as the lower concentrations. The 0.1
and 0.01 concentrations are not significantly different
(p=0.11).
[0091] FIG. 16 shows fire ant worker mortality is shown for 1.0,
0.1, and 0.01% concentrations of the tyramine derivative
N-methyl-tyramine (HCl). All three concentrations are significantly
different from the sucrose control. These results are important
because the cost of the HCl salt is less than the free base, making
it potentially a more cost-effective option for commercialization.
Also, significant mortality over 100-fold dilution is a key
characteristic of an effective fire ant bait insecticide. The 1.0%
and 0.1% concentrations are significantly different from the 0.01
concentration (p<0.0001). The 1% and 0.1% are not significantly
different (p=0.63). The N-methyl tyramine HCl appears to be quicker
acting compared to the N-methyl tyramine free-base. FIGS. 17, 18,
and 19 use N-methyl tyramine HCl for comparisons with tyramine at
the same concentrations, due to the advantages noted above. The
results show greater mortality for the N-methyl derivative than the
tyramine at the same concentrations (see details below). In
contrast, fire ant worker feeding experiments with 1%
N,N-dimethyl-tyramine showed no difference in mortality (results
not shown) from the 10% sucrose control (P=0.97). Therefore, the
addition of another methyl group on the nitrogen negated the worker
mortality activity observed with tyramine and N-methyl-tyramine.
This suggests that the added steric hindrance of a second methyl
group prevents its interaction with one or more critical
receptors.
[0092] FIG. 17 shows a comparison of the effects of 1.0%
concentrations of tyramine and 1.0% N-methyl tyramine (HCl) versus
the 10% sucrose control. The 1.0% N-methyl tyramine (HCl) shows
significantly faster worker mortality than the 1.0% tyramine
(p<0.001). Mortality results for both treatments are
significantly different from the sucrose control.
[0093] FIG. 18 shows a comparison of the effects of 0.1%
concentrations of tyramine and N-methyl tyramine (HCl) versus the
10% sucrose control. Mortality results for both treatments are
significantly different from the sucrose control. Fire ant worker
mortality results for 0.1% N-methyl tyramine (HCl) are not
significantly faster than the 0.1% tyramine (p=0.22).
[0094] FIG. 19 shows a comparison between 0.01% concentrations of
tyramine and 0.01% N-methyl tyramine (HCl) versus the 10% sucrose
control. Mortality results for both treatments are significantly
different from the sucrose control. Fire ant worker mortality for
0.01% N-methyl tyramine (HCl) is significantly faster than the
0.01% tyramine (p<0.01). This result further supports the
advantage of the N-methyl tyramine HCl over the free base.
[0095] FIG. 20 shows fire ant mortality results for two
concentrations (1.0 and 0.1% in 10% sucrose) of
3-chloro-4-methoxyphenethylamine. Both treatment concentrations
were significantly different from the 10% sucrose control.
Mortality for the 0.1% concentration was not significantly greater
than that for the 1.0% concentration (p=0.15). While the results
were significant, the mortality (about 8%) was not high enough to
warrant further testing.
[0096] FIG. 21 shows worker mortality results for tyramine
derivatives when the 4-hydroxyl group is replaced by a halogen.
Significant, but low, worker mortality occurs for 4-chloro-,
4-bromo-, and 4-flurophenethylamine. All concentrations were at
1.0% in 10% sucrose solution. There are no differences between the
three halogen derivatives (p=0.62). In contrast, fire ant worker
mortality was measured (data not shown) for 1% concentrations of
the three possible 2- or 3- or 4-methoxy-phenethylamine (tyramine)
derivatives. No difference in mortality (p=0.12) was observed when
compared to 10% sucrose control (data not shown).
[0097] FIGS. 22 and 23 show two physiological events: (i) ovariole
development and (ii) production of a worker attractant pheromone.
Both events occur when female sexuals (alates) are removed from
their mother colony and out of the influence of the colony queen.
Under these conditions female alates will lose their wings and
start ovariole development and pheromone production. The alates in
treatments and controls were monitored once every 24 h for loss of
wings (dealation). The ovarioles and poison sacs from dealates were
immediately dissected. The poison sacs were immersed in hexane (30
.mu.l) and placed in a vial and kept frozen until used in the queen
pheromone bioassay (see below). Digital pictures of the ovarioles
were taken directly after dissection for subsequent quantification
by measuring the planar area of each of the two ovariole clusters
(Leica Application Suite 4.4.02, Leica Microsystem (Switzerland),
with an interactive measurement module). The level of worker
attraction to poison sac extracts from dealates was quantified
using a procedure modified from Lofgren, et al. 1983 (Annals ESA.
1983; 76:44-50).
[0098] FIG. 22 shows a comparison of planar ovariole area (pixels)
measurements of treatment and control sub-colonies (N=15/treatment
or control) composed of alates with workers, but no queen, after
being fed the indicated treatments/control. Control was 10% sucrose
and the treatments were 1% tyramine or 1% N-methyl-tyramine, or 1%
Di-methyl-tyramine. Bars with the same letter above them are not
significantly different (p>0.05), whereas they are significantly
different (p<0.05) if the letters are different. Therefore,
results for tyramine and N-Methyl tyramine are not different from
each other, but are different from dimethyl tyramine and the
sucrose control.
[0099] FIG. 23 shows results from a bioassay that measured
attraction of workers to female sexual poison gland extracts. Bars
with the same letter above them are not significantly different
(p>0.05), whereas they are significantly different (p<0.05)
if the letters are different. Therefore, poison sac extracts from
N-methyl tyramine treatments induced significantly greater worker
attraction than poison sac extracts from tyramine treatments;
however, worker attraction to poison sac extracts from tyramine,
tyramine HCl, and N-methyl tyramine treatments were significantly
different from those from the N,N-dimethyl tyramine treatment and
the sucrose and hexane controls. The tyramine HCl was not different
from tyramine or N-methyl tyramine because of its greater replicate
variance. N-methyl tyramine showed low variance and the highest
worker response.
EXAMPLE 15
[0100] FIGS. 24A and 24B show results for the sucrose (10%) control
and two treatments, tyramine (1%) and N-methyl-tyramine (1%), both
dissolved in 10% sucrose. The control and test colonies were
monogyne queenright colonies, containing a single queen, brood and
workers started in the laboratory from newly mated queens collected
in the field. The mean number of workers per colony was
8,400.+-.400. FIG. 24A shows the % worker mortality at the end of
the experiment (27 d). FIG. 24B shows queen mortality results at
the end of the experiment. Bars with the same letter above them are
not significantly different (p>0.05), whereas they are
significantly different (p<0.05) if the letters are different.
Therefore, for both worker and queen mortality, the results for
tyramine and N-methyl tyramine are not different from each other,
but are significantly different from the corresponding controls.
Previous experiments used queenless sub-colonies, rather than the
queenright colonies in these experiments. The mortality effect on
colony queens was unexpected, since worker fire ants are completely
sterile and cannot reproduce even if the queen dies. Therefore,
when the reproductive queen dies no more brood is produced and the
colony is no longer functional--it is considered dead. For N-methyl
tyramine all queen replicates (5) died; therefore, 100% of the
colonies were considered dead. For tyramine one colony queen
survived out of five. Therefore, 80% of the treated colonies were
considered dead.
[0101] The results demonstrate the surprising and unexpected
negative effect of tyramine and its derivatives on of fire ants.
While the dosages used are significantly higher than naturally
found within colonies, the highly deleterious effects on fire ant
colonies when delivered at such dosage levels was surprising and
unexpected. These compounds are not known to have deleterious
effects on insects. The expectations of one skilled in the art
would not include such predictions, particularly when coupled with
known properties of the biogenic amines and the differences
observed among the biogenic amines tested and the absence of effect
on a different ant species.
[0102] While this invention may be embodied in many different
forms, there are described in detail herein specific preferred
embodiments of the invention. The present disclosure is an
exemplification of the principles of the invention and is not
intended to limit the invention to the particular embodiments
illustrated. All patents, patent applications, scientific papers,
and any other referenced materials mentioned herein are
incorporated by reference in their entirety. Furthermore, the
invention encompasses any possible combination of some or all of
the various embodiments and characteristics described herein and/or
incorporated herein. In addition the invention encompasses any
possible combination that also specifically excludes any one or
some of the various embodiments and characteristics described
herein and/or incorporated herein.
[0103] The amounts, percentages and ranges disclosed herein are not
meant to be limiting, and increments between the recited amounts,
percentages and ranges are specifically envisioned as part of the
invention. All ranges and parameters disclosed herein are
understood to encompass any and all subranges subsumed therein, and
every number between the endpoints. For example, a stated range of
"1 to 10" should be considered to include any and all subranges
between (and inclusive of) the minimum value of 1 and the maximum
value of 10 including all integer values and decimal values; that
is, all subranges beginning with a minimum value of 1 or more,
(e.g., 1 to 6.1), and ending with a maximum value of 10 or less,
(e.g. 2.3 to 9.4, 3 to 8, 4 to 7), and finally to each number 1, 2,
3, 4, 5, 6, 7, 8, 9, and 10 contained within the range.
[0104] Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as molecular weight,
reaction conditions, and so forth as used in the specification and
claims are to be understood as being modified in all instances by
the term "about." Accordingly, unless otherwise indicated, the
numerical properties set forth in the following specification and
claims are approximations that may vary depending on the desired
properties sought to be obtained in embodiments of the present
invention. As used herein, the term "about" refers to a quantity,
level, value, or amount that varies by as much as 30%, preferably
by as much as 20%, and more preferably by as much as 10% to a
reference quantity, level, value, or amount.
[0105] Other embodiments of the invention will be apparent to those
skilled in the art from a consideration of this specification or
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with
the true scope and spirit of the invention being indicated by the
following claims. Although any methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the present invention, the preferred methods and
materials are herein described. Those skilled in the art may
recognize other equivalents to the specific embodiments described
herein which equivalents are intended to be encompassed by the
claims attached hereto.
* * * * *